Collagen hydrolysate composition for relieving joint pain after exercise

Abstract

The present invention relates to a composition comprising chondroitin sulfate and collagen hydrolysate, the composition being useful for alleviating pain, preferably joint pain after exercise, and a method for preparing the composition by hydrolyzing a collagen-containing substance comprising different types of collagen.

Description

Technical Field

[0001] The present invention relates to a composition for improving pain such as joint pain, etc. In addition, the present invention relates to a method for preparing the composition. Background Art

[0002] Joint pain is a common condition that can occur in middle-aged or older adults. Joint pain can have different causes, but the most common causes are related to heavy exercise, injury, or an underlying medical condition such as obesity or degenerative joint disease.

[0003] Joint pain is most often observed in joints that are subjected to heavy and frequent loads, i.e., mainly in the knee joint, shoulder joint, and hip joint. These joints are fully innervated by sensory nerves and sympathetic nerves. The movement of the joint stimulates free nerve endings, leading to mechanical sensation of pain. In arthritic joints, so-called "silent nociceptors" also become active, thereby further promoting the generation of joint pain (McDougall et al. Arthritis Res. Ther. 8, 220, 2006).

[0004] Joint pain can be observed in healthy and physically active individuals. Joint pain can also be observed under pathological conditions. In healthy individuals, joint pain usually aggravates (flourish) during a few days to a few weeks after a lot of or prolonged exercise, after which the pain can disappear naturally. This can be considered as acute joint pain. It is believed that (short-term) acute joint pain after a lot of or prolonged exercise events is caused by the limited ability of low friction movement in the joint and higher load transmission. Under pathological conditions, joint pain is typically chronic (i.e., it will not disappear naturally). For example, the catabolic changes in the joints seen in osteoarthritis lead to chronic pain and joint stiffness. Therefore, the relevant inflammation in the joint can cause peripheral sensitization and joint pain, and has nothing to do with excessive load or exercise events. It is believed that osteoarthritis cannot be cured at present, and therefore continuous symptomatic treatment of pain is needed.

[0005] Joint pain is currently treated with conventional drugs to relieve pain and inflammation. Conventional drugs for joint pain include simple analgesics (e.g., acetaminophen) and nonsteroidal anti-inflammatory drugs. These conventional drugs have several limitations. First, these drugs cannot regulate the underlying pathology of the disease, which is necessary for effective and long-term pain relief. Second, these drugs often cause adverse effects, such as gastrointestinal toxicity, cardiovascular problems, kidney and liver damage, and rash (Zhang et al. Osteoarthr. Cartil., 18 (4), 2010, 476–499, Sing et al. Arthritis Res. Ther., 2006, 8 (5), R153). Third, the risk of adverse effects of these conventional drugs makes them unsuitable for treating healthy individuals and physically active individuals (i.e., as non-therapeutic uses), such as in acute joint pain.

[0006] Based on previous studies, safer and more readily available alternatives are needed for the management of joint pain. A widely studied dietary supplement is glucosamine sulfate (GS). GS is a sulfated amino monosaccharide that serves as a substrate in the biosynthesis of cartilage proteoglycans. GS is believed to relieve joint pain by inhibiting catabolic enzymes and proinflammatory cytokines. Although GS is currently used to treat joint pain, systematic reviews have shown that GS may have only a small to moderate effect in alleviating joint pain (Knapik et al. J. Spec. Oper. Med., Winter 2018; 18(4): 139-147).

[0007] Other dietary supplements that have been studied include combinations of active ingredients found in cartilage (and its derivatives), including combinations of collagen and chondroitin sulfate. For example, Ma et al. (Journal of Functional Foods (2021), 78, 104376) US2007293427A1 and US2003091652A1 describe compositions for preventing osteoarthritis and related symptoms, which include collagen hydrolysates and chondroitin sulfate primarily derived from type II collagen. JP2009051833A discloses a composition for improving joint pain, comprising a combination of collagen, methylsulfonylmethane (MSM), glucosamine, and chondroitin sulfate.

[0008] There is still an unmet need for compositions that are more effective and / or safer for improving joint pain. In particular, there is an unmet need for compositions that are more effective and / or safer for improving joint pain in healthy individuals after exercise. The present invention aims to provide such compositions. Summary of the Invention

[0009] The present inventors have discovered a method for obtaining a composition based on collagen hydrolysate and chondroitin sulfate, the use of which has a high efficacy in alleviating joint pain. The composition of the present invention was found to be superior to other compositions based on collagen hydrolysate and chondroitin sulfate in alleviating joint pain.

[0010] For example, compared to compositions intended to treat a broad range of diseases (i.e., osteoarthritis and related joint diseases), the compositions of the present invention most fundamentally contain more appropriate amounts of active ingredients that maximize pain relief. Furthermore, the compositions of the present invention effectively alleviate pain in a wide range of subject groups, including both chronic and acute pain, and / or both pathological and physically active subjects.

[0011] The present inventors have found that the compositions of the present invention have characteristic features which, individually and particularly in combination, achieve an unexpectedly high degree of relief of joint pain. The present inventors have found that more effective relief of joint pain is achieved with compositions in which the collagen hydrolysate is derived from a collagen-containing starting material comprising a mixture of type I and type II collagen. In particular, the collagen hydrolysate produced from a collagen-containing starting material comprising type I collagen, type II collagen and type III collagen in a specific weight ratio has an improved pain-reducing effect compared to other compositions comprising collagen hydrolysate and chondroitin sulfate. The compositions of the present invention are more effective in alleviating pain than compositions containing primarily type I collagen or type II collagen, respectively.

[0012] The collagen hydrolysate of the present invention may be characterized by a hydroxyproline / hydroxylysine molar ratio (Hyp / Hyl ratio) of 8 to 15 (i.e., representing a mixture of peptides derived from appropriate amounts of different types of collagen); and / or an average molecular weight of greater than 1000 Da and less than 10,000 Da. The Hyp / Hyl ratio and / or molecular weight have been shown to be relevant characteristics for achieving a high degree of pain relief. Furthermore, the present inventors have discovered that a high degree of joint pain relief can be achieved with a composition comprising approximately 70% collagen hydrolysate and approximately 30% chondroitin sulfate.

[0013] In one aspect, the present invention relates to a composition comprising 10% to 40% by weight of chondroitin sulfate and 60% to 90% by weight of collagen hydrolysate, calculated based on the total weight of chondroitin sulfate and collagen hydrolysate.

[0014] Collagen hydrolysate can be obtained from the hydrolysis of collagen-containing substances.

[0015] The collagen-containing material preferably contains 40% to 60% by weight of type I collagen, calculated based on the total weight of collagen in the collagen-containing material.

[0016] The collagen-containing material preferably contains 40% to 60% by weight of type II collagen, calculated based on the total weight of collagen in the collagen-containing material.

[0017] The collagen-containing substance preferably comprises 5% to 20% by weight of type III collagen, calculated on the total weight of collagen in the collagen-containing substance.

[0018] In one aspect, the present invention relates to a composition comprising 10% to 40% by weight of chondroitin sulfate and 60% to 90% by weight of collagen hydrolysate, calculated based on the total weight of chondroitin sulfate and collagen hydrolysate.

[0019] The collagen hydrolysate preferably contains 40% to 60% by weight of collagen derived from type I collagen, calculated based on the total weight of collagen in the collagen hydrolysate.

[0020] The collagen hydrolysate preferably contains 40% to 60% by weight of collagen derived from type II collagen, calculated based on the total weight of the collagen in the collagen hydrolysate, and the collagen hydrolysate preferably further contains 5% to 20% by weight of collagen derived from type III collagen, calculated based on the total weight of the collagen in the collagen hydrolysate.

[0021] In one aspect, the present invention relates to the use of a composition as disclosed herein in therapeutic treatment.

[0022] In one aspect, the present invention relates to the use of a composition as disclosed herein for the non-therapeutic improvement of pain, preferably for the non-therapeutic improvement of joint pain, for example after exercise.

[0023] In one aspect, the present invention relates to the use of a composition as disclosed herein for the therapeutic improvement of pain, preferably for the therapeutic improvement of joint pain, eg after exercise.

[0024] In one aspect, the invention relates to a method of treating pain in a subject, preferably joint pain, such as after exercise, comprising administering to the subject a composition as disclosed herein.

[0025] In one aspect, the present invention relates to the use of a composition as disclosed herein for the preparation of a medicament for ameliorating pain, preferably joint pain, for example after exercise. DETAILED DESCRIPTION

[0026] Some embodiments of the compositions of the present invention

[0027] In one embodiment, the present invention relates to a composition comprising 10% to 40% by weight of chondroitin sulfate and 60% to 90% by weight of collagen hydrolysate, calculated based on the total weight of chondroitin sulfate and collagen hydrolysate.

[0028] Chondroitin sulfate can be present in the composition in an amount of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, calculated based on the total weight of the chondroitin sulfate and collagen hydrolysate. Chondroitin sulfate can be present in the composition in an amount of no more than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%, calculated based on the total weight of the chondroitin sulfate and collagen hydrolysate.

[0029] In one embodiment, the chondroitin sulfate and / or collagen hydrolysate as disclosed herein is derived from a collagen-containing substance, preferably a collagen-containing substance subjected to (enzymatic) hydrolysis. In one embodiment, the chondroitin sulfate and / or collagen hydrolysate is derived from the same collagen-containing substance, wherein the collagen-containing substance is preferably subjected to (enzymatic) hydrolysis.

[0030] In one embodiment, the collagen hydrolysate disclosed herein contains 1 wt % to 25 wt % (e.g., 2 wt % to 20 wt %, or 4 wt % to 15 wt %, or 5 wt % to 10 wt %) collagen derived from type III collagen, based on the total weight of collagen in the collagen hydrolysate.

[0031] Substances containing collagen

[0032] The collagen-containing material as disclosed herein is preferably composed of one or more (animal) collagen-containing tissues, although it is not excluded that the collagen-containing material is (additionally) prepared by introducing purified collagen products such as (commercially available) collagen powder.

[0033] The preferred amount of collagen subtype (for example, type I, type II, type III collagen) in the material containing collagen can be achieved by selecting and combining suitable tissue or the material containing collagen. Technicians know that the relative amount of collagen subtype depends on the source of tissue and tissue (such as animal species, age of animal). In the art, the amount and type of collagen are known, or textbooks and other common methods such as ELISA (or similar antibody-based) can be used to determine. In order to control and determine the actual collagen content and the collagen subtype in the material containing collagen used in the present invention, such qualitative and quantitative methods are preferably used. According to outcome, technicians can adjust the method for processing the material containing collagen and / or combining two or more materials containing collagen.

[0034] In one embodiment, the collagen-containing material is derived from (animal) tissue, wherein the relative amounts of collagen subtypes naturally present are within the desired range, preferably 40 wt% to 60 wt% collagen type I and 40 wt% to 60 wt% collagen type II, calculated based on the total weight of collagen in the collagen-containing material.

[0035] In one embodiment, the collagen-containing substance is a combination of two or more (animal) tissues, such as collagen subtypes (e.g., type I, type II, type III collagen) with different relative amounts. For example, a first collagen-containing substance comprising X weight % type I collagen (but lacking type II collagen) can be combined with a second collagen-containing substance comprising X weight % type II collagen (but lacking type I collagen) in a weight ratio of 1:1 to obtain a collagen-containing substance comprising 50 weight % type I collagen and 50 weight % type II collagen, calculated based on the total weight of the collagen in the collagen-containing substance. For collagen-containing substances comprising a mixture of collagen subtypes (e.g., both type I and type II), the amount of the desired first and additional collagen-containing substances can be appropriately weighted.

[0036] Depending on the type of (animal) tissue, a selected tissue region may be more suitable for achieving the composition of the collagen-containing substance of the present invention. For example, it is known that the sternum contains both bone and cartilage tissue. By obtaining appropriate regions or layers of tissue, the relative amounts of type I collagen (i.e., relatively high in bone tissue) and type II collagen (i.e., relatively high in cartilage tissue) can be adjusted. In addition, such processing can achieve an increased purity of a certain type of collagen.

[0037] There are different suitable methods for quantitatively measuring the content of total collagen and collagen subtypes in (tissue) samples. Collagen determination based on Sirius Red is a widely used determination for collagen quantification, and it is based on the precipitation of soluble or dissolved collagen. Sirius Red is known to bind to fibrous collagen. Collagen determination based on Sirius Red is commercially available (e.g., Chondrex Inc Sirius Red Total Collagen Detection Assay Kit, Biocolor Sircol assay, QuickZyme Soluble Collagen assay). In the hydrolyzed sample, the collagen content is preferably measured using a hydroxyproline-based collagen assay, which is also commercially available (e.g., Chondrex Inc Hydroxyproline Assay Kit, QuickZyme Total Collagen assay). The hydroxyproline assay is suitable for determining the total collagen content of both native and denatured collagen.

[0038] The preferred method for quantifying collagen subtypes in a sample preferably includes using specific antibodies to detect collagen, and subsequently quantifying the amount of the bound antibody. The amount of collagen can be calculated using a standard curve using a known amount of collagen. Collagen type-specific antibodies are provided by companies such as Chondrix Inc, R&D Systems, and Abcam. It is generally believed that the method based on enzyme-linked immunosorbent assay (ELISA) is very accurate in quantifying the antibody level in a given sample. ELISA is one of the most commonly used biochemical assays, and technicians can set up (their own) suitable ELISA protocols based on target protein and / or sample. Commercial ELISA kits can also be used to determine the amount of collagen subtypes (e.g., type I collagen, type II collagen, type III collagen) in a sample. For example, Chondrex Inc provides ELISA kits for measuring bovine type I collagen (catalog #6014), pig (catalog #6015), and ELISA kits for measuring multispecies type II collagen (catalog #6018).

[0039] A preferred method for determining collagen subtypes in hydrolyzed samples is by sequencing collagen subtype-specific amino acid sequences via liquid chromatography with tandem mass spectrometry (LC-MS / MS), for example based on the method of Piestansky et al. (Pharm.Biomed.Anal.2020Sep 10; 189: 113449). A preferred approach is the following, which is disclosed by Ma et al. (Journal of Functional Foods (2021), 78, 104376): The sample was chromatographed on a C18 column (2.1 mm × 100 mm, 1.7 μm) at 40 ° C using a TripleTOF 5600 system (AB SCIEX Corporation Co., Ltd, USA). Mobile phase A was 0.1% formic acid in acetonitrile, and mobile phase B was 0.1% formic acid in water. The flow rate and injection volume were 0.25 mL / min and 10 μL. Elution conditions were set to 0 to 39 minutes, 5% to 80% A; 42 minutes, 80% A; 42.5 minutes, 80% to 5% A; 46 minutes, 5% A. The mass spectrometer scan range was 350 to 1500 m / z, using positive ion reaction mode, and the data were retrieved by ProteinPlot™ (version 5.0.2.0) software using the uniprot collagen database. The false discovery rate (FDR) was set to FDR≤0.01.

[0040] In a preferred embodiment, the present invention relates to a composition comprising 10% to 40% by weight of chondroitin sulfate and 60% to 90% by weight of collagen hydrolysate, calculated on the total weight of chondroitin sulfate and collagen hydrolysate.

[0041] Collagen hydrolysate can be obtained from the hydrolysis of collagen-containing substances.

[0042] The collagen-containing substance preferably comprises 40% to 60% by weight of type I collagen, calculated based on the total weight of collagen in the collagen-containing substance, and / or

[0043] The collagen-containing substance preferably comprises 40% to 60% by weight of type II collagen, calculated based on the total weight of collagen in the collagen-containing substance, and / or

[0044] The collagen-containing substance preferably comprises 5% to 20% by weight of type III collagen, calculated on the total weight of collagen in the collagen-containing substance.

[0045] In one embodiment, the present invention relates to a composition comprising 10% to 40% by weight of chondroitin sulfate and 60% to 90% by weight of collagen hydrolysate,

[0046] The collagen hydrolysate preferably comprises 40% to 60% by weight of type I collagen, calculated based on the total weight of collagen in the collagen hydrolysate, and / or

[0047] The collagen hydrolysate preferably comprises 40% to 60% by weight of type II collagen, calculated based on the total weight of collagen in the collagen hydrolysate, and / or

[0048] The collagen hydrolysate preferably further comprises 5 wt% to 20 wt% of type III collagen, calculated based on the total weight of collagen in the collagen hydrolysate.

[0049] Hyaluronic acid is a component of cartilage, which has been suggested for the treatment of joint pain (Migliore et al. Rheumatol. Ther. 2021 Dec; 8 (4): 1617-1636). Because hyaluronic acid is a natural component of cartilage tissue, compositions based on collagen hydrolysate (and / or chondroitin sulfate) also typically include a large amount of hyaluronic acid (typically at least 0.5 wt %). The presence of hyaluronic acid can be desirable or undesirable (pollution). Unexpectedly, the present inventors found that compared with compositions comprising 0.5 wt % or more hyaluronic acid, compositions comprising less than 0.5 wt % hyaluronic acid provide improved relief of joint pain. This means that it is preferred to have relatively little hyaluronic acid in the composition, and / or a high weight ratio of chondroitin sulfate: hyaluronic acid.

[0050] In one embodiment, the compositions as disclosed herein comprise less than 2% by weight hyaluronic acid, preferably less than 1% by weight hyaluronic acid, more preferably less than 0.5% by weight hyaluronic acid, even more preferably less than 0.1% by weight hyaluronic acid, based on the weight of the composition. For example, the compositions as disclosed herein may comprise hyaluronic acid in an amount of 0% to 1% by weight, or 0% to 0.5% by weight, or 0% to 0.1% by weight, or 0% to 0.05% by weight, or 0% to 0.01% by weight, based on the weight of the composition.

[0051] In one embodiment, the weight ratio of chondroitin sulfate to hyaluronic acid in the composition as disclosed herein is 10,000: 1 to 10: 1, preferably 1,000: 1 to 100: 1. The weight ratio of chondroitin sulfate to hyaluronic acid in the composition as disclosed herein may be at least 10: 1, or at least 100: 1, or at least 1,000: 1, or at least 10,000: 1. Additionally or alternatively, the weight ratio of chondroitin sulfate to hyaluronic acid in the composition as disclosed herein may not exceed 10,000: 1, or not exceed 1,000: 1, or not exceed 100: 1, or not exceed 10: 1.

[0052] The molecular weight of "hyaluronic acid" as disclosed herein is preferably 4000 to 10000 kDa, preferably 5000 to 9000 kDa, more preferably 6000 to 8000 kDa, wherein the molecular weight is preferably an average molecular weight.

[0053] Some embodiments of chondroitin sulfate of the present invention

[0054] The term "chondroitin sulfate" (i.e., chondroitin sulfate) as used herein Chondroitin sulfate) refers to a negatively charged polymeric glycosaminoglycan composed of alternating glucuronic acid and N-acetylhexosamine residues linked by (β1-3 hexuronic acid and β1-4-N-acetylhexosamine bonds. The term "chondroitin sulfate" herein encompasses fractions with little or no sulfation. The term "chondroitin sulfate" is used interchangeably herein with the term "chondroitin." A preferred method for quantifying chondroitin sulfate in a substance is by enzymatic hydrolysis of chondroitin sulfate followed by high performance liquid chromatography. For example, chondroitin sulfate can be treated with chondroitinase ABC or chondroitinase AC enzymes to selectively hydrolyze chondroitin sulfate into unsaturated disaccharide units. Chondroitinase ABC will hydrolyze both chondroitin sulfate and dermatan sulfate (sometimes referred to as "chondroitin sulfate B"), while chondroitinase AC is specific for chondroitin sulfate. The resulting unsaturated disaccharide units (Ji) can then be separated and quantified by ion exchange chromatography or reverse phase chromatography under UV detection, conductivity detection, or pre-column derivatization with fluorescence detection. et al. Journal of AOAC INTERNATIONAL, Vol. 90, No. 3, May 1, 2007, pp. 659-669). A preferred embodiment is disclosed by Ji et al. (Journal of AOAC INTERNATIONAL, Vol. 90, No. 3, May 1, 2007, pp. 659-669).

[0055] The average molecular weight of chondroitin sulfate as disclosed herein may be at least 1 kDa, or at least 2.5 kDa, or at least 5 kDa, or at least 7.5 kDa, or at least 10 kDa, or at least 12.5 kDa, or at least 15 kDa, or at least 17.5 kDa, or at least 20 kDa, or at least 25 kDa, or at least 30 kDa. Additionally or alternatively, the average molecular weight of chondroitin sulfate as disclosed herein may be no more than 40 kDa, or no more than 35 kDa, or no more than 30 kDa, or no more than 25 kDa, or no more than 20 kDa, or no more than 17.5 kDa, or no more than 15 kDa, or no more than 12.5 kDa, or no more than 10 kDa.

[0056] In a preferred embodiment, the average molecular weight of chondroitin sulfate as disclosed herein is from 10 to 35 kDa, preferably from 15 to 30 kDa, more preferably from 17 to 27 kDa.

[0057] The present inventors have discovered that the molecular weight of chondroitin sulfate may determine its biological activity in alleviating pain, particularly in combination with collagen hydrolysate.

[0058] In one embodiment, a composition comprising chondroitin sulfate having an average molecular weight of 15 to 30 kDa provides greater pain relief compared to a composition comprising chondroitin sulfate having an average molecular weight of less than 15 kDa or greater than 30 kDa.

[0059] In one embodiment, a composition comprising chondroitin sulfate having an average molecular weight of 17 to 27 kDa provides greater pain relief compared to a composition comprising chondroitin sulfate having an average molecular weight of less than 17 kDa or greater than 27 kDa.

[0060] A suitable and preferred method for measuring the molecular weight of chondroitin sulfate is by gel permeation chromatography (GPC) or high performance size-exclusion chromatography (HP-SEC), for example after treating a sample containing chondroitin sulfate with a sodium acetate-ethanol solution to precipitate glycosaminoglycans and obtain a fraction (precipitate) rich in chondroitin sulfate. In addition or alternatively, a preferred method for determining the molecular weight of chondroitin sulfate is by GPC as described by Vázquez et al. (Polymers (Basel). 2020 Nov 6; 12(11): 2613).

[0061] Some embodiments of the collagen hydrolyzate of the present invention

[0062] The term "collagen hydrolysate" as used herein means a mixture of short amino acid chains of collagen-containing materials with native (full-length) collagen, preferably obtained by a hydrolysis step, including enzymatic hydrolysis (enzymatic hydrolysis) (also referred to as enzymatic hydrolysis). "Collagen hydrolysate" herein can encompass collagen that has been hydrolyzed or partially hydrolyzed. The term "collagen hydrolysate" can be used interchangeably with the term "collagen hydrolysate," "hydrolyzed collagen," or "collagen peptides" and is synonymous. "Collagen hydrolysate" herein can be produced in a one-step process from a collagen-containing material, or by an intermediate gelatin stage, wherein preferably, for example, type A and / or type B gelatin (e.g., a mixture of type A and type B gelatin) is used. "Collagen hydrolysate" herein can encompass hydrolyzed gelatin, which is obtained by the hydrolysis of gelatin (obtained from collagen). The terms "collagen hydrolysate," "gelatin hydrolysate," "hydrolyzed gelatin," and "hydrolysed gelatin" are used interchangeably and synonymously in this disclosure.

[0063] In one embodiment, the average molecular weight of the collagen hydrolysate as disclosed herein is 500 to 10,000 Da, for example 600 to 9,000 Da, or 700 to 8,000 Da, or 800 to 7,000 Da, or 900 to 6,000 Da, or 1,000 to 7,000 Da, or 1,100 to 6,000 Da, or 1,200 to 5,000 Da, or 1,300 to 6,000 Da, or 1,400 to 5,000 Da, or 1,500 to 4,500 Da.

[0064] The average molecular weight as disclosed herein is preferably a weight average molecular weight.

[0065] It was shown that pain relief was greatest when the average molecular weight of the collagen hydrolysate was greater than 1000 Da and less than 10,000 Da. This effect was most pronounced for collagen hydrolysates with an average molecular weight greater than 1000 Da and less than 5000 Da.

[0066] In one embodiment, the average molecular weight of the collagen hydrolysate as disclosed herein is from 1000 to 10000 Da.

[0067] In one embodiment, the average molecular weight of the collagen hydrolysate as disclosed herein is from 1000 to 5000 Da, preferably from 1100 to 5000 Da, or from 1200 to 4500 Da, or from 1300 to 4000 Da, or from 1400 to 3500 Da, or from 1500 to 3000 Da.

[0068] A suitable and preferred method for measuring the molecular weight of collagen hydrolysate is by high performance size exclusion chromatography (HP-SEC), for example after treating a sample containing collagen hydrolysate with a sodium acetate-ethanol solution to precipitate a protein-deficient fraction (comprising glycosaminoglycans), and performing HP-SEC of the collagen hydrolysate using the protein-rich fraction (supernatant). In addition or in lieu thereof, a preferred method for determining the molecular weight of collagen hydrolysate is according to Edgar et al. (Sci. Rep. 2018 Jul 11; 8(1): 10474).

[0069] In one embodiment, the collagen hydrolysate as disclosed herein comprises hydroxyproline in an amount of 1 wt% to 30 wt%, preferably 2 wt% to 20 wt%, more preferably 5 wt% to 15 wt%, most preferably 8 wt% to 12 wt%, all based on the total weight of amino acids in the collagen hydrolysate.

[0070] In one embodiment, the collagen hydrolysate as disclosed herein comprises hydroxylysine in an amount of 0.1 wt% to 5 wt%, preferably 0.2 wt% to 2 wt%, more preferably 0.5 wt% to 1.25 wt%, most preferably 0.7 wt% to 1 wt%, all based on the total weight of amino acids in the collagen hydrolysate.

[0071] In one embodiment, the collagen hydrolysate as disclosed herein comprises hydroxyproline in an amount of 10 to 200 amino acids (ie AAs) per 1000 AAs in the collagen hydrolysate, preferably 50 to 100 AAs per 1000 AAs in the collagen hydrolysate.

[0072] In one embodiment, the collagen hydrolysate as disclosed herein comprises hydroxylysine in an amount of 1 to 20 AAs per 1000 AAs in the collagen hydrolysate, preferably 2 to 10 AAs per 1000 AAs in the collagen hydrolysate.

[0073] In one embodiment, the collagen hydrolysate as disclosed herein comprises hydroxyproline and hydroxylysine in a molar ratio (hydroxyproline:hydroxylysine) of 1 to 30 (e.g., 5 to 19, or 6 to 18, or 7 to 17, or 8 to 16, or 9 to 15, or 10 to 14, or 11 to 13), preferably 5 to 20, more preferably greater than 8 and less than 15.

[0074] Collagen subtypes differ significantly in their hydroxyproline / hydroxylysine molar ratios, which can be used to identify them. The hydroxyproline / hydroxylysine molar ratio in a (hydrolyzed) sample can be correlated with the collagen subtype in the collagen-containing starting material (Ignat'eva et al. Journal of Analytical Chemistry, 2007, Vol. 62, No. 1, pp. 51–57, Blumenkrantz et al. Clinical Biochemistr. Vol. 13, No. 4, August 1980, pp. 177–183). In general:

[0075] A hydroxyproline / hydroxylysine ratio of <7.5 (e.g., about 5) indicates that (in the collagen-containing starting material) type II collagen is predominant;

[0076] A hydroxyproline / hydroxylysine ratio > 10 (e.g. 10 to 20) indicates (in the collagen-containing starting material) that type I collagen is predominant;

[0077] A hydroxyproline / hydroxylysine ratio of >15 (e.g. 15 to 25) indicates (in the collagen-containing starting material) that type III collagen is predominant;

[0078] A hydroxyproline / hydroxylysine ratio > 8 and < 15 indicates (in the collagen-containing starting material) a mixture of collagen types I, II and III.

[0079] The above correlation between the hydroxyproline / hydroxylysine ratio and collagen subtypes is used in the present disclosure.

[0080] In one embodiment, a hydroxyproline / hydroxylysine molar ratio of greater than 8 and less than 15 indicates that 40% to 60% by weight of the collagen is derived from type I collagen, and 40% to 60% by weight of the collagen is derived from type I collagen, and further preferably 40% to 60% by weight of the collagen is derived from type II collagen, wherein the weight % is calculated based on the total weight of the collagen (e.g., the collagen in the collagen-containing material subjected to hydrolysis).

[0081] A preferred method for determining the molar ratio of hydroxyproline to hydroxylysine is by reverse phase high performance liquid chromatography and mass spectrometry, such as described by Langrock et al. (Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2007 Mar. 1; 847(2): 282-8). Hydroxyproline can also be measured using a hydroxyproline-based collagen assay as disclosed herein. A preferred method for determining the molar ratio of hydroxyproline to hydroxylysine is according to the publication by Blumenkrantz et al. (Clinical Biochemistr. Vol. 13, No. 4, August 1980, pp. 177-183).

[0082] Method for preparing the composition

[0083] In one embodiment, one or more components of the composition (eg collagen hydrolysate, chondroitin sulfate) are obtained by hydrolyzing, preferably enzymatically hydrolyzing, a collagen-containing material.

[0084] The enzymatic hydrolysis as disclosed herein preferably comprises subjecting the collagen-containing material to one or more enzymes selected from the group consisting of serine proteases, alkaline proteases, neutral proteases, flavor proteases, complex proteases, thiol proteases, bromelain, metalloproteases, aspartame, proteases, carboxypeptidases, pepsin, chymotrypsin, trypsin, cathepsin K, chymotrypsin, papain and subtilisin.

[0085] In one embodiment, the collagen-containing material as disclosed herein is derived from a tissue comprising cartilage. In one embodiment, the collagen-containing material as disclosed herein is derived from a tissue comprising bone. In one embodiment, the collagen-containing material as disclosed herein is derived from a tissue comprising bone and cartilage.

[0086] "Cartilage" as disclosed herein is preferably hyaline cartilage.

[0087] As used herein, substance Y is said to be "derived from" substance X if it comprises substance X. Additionally or alternatively, as used herein, substance Y is said to be "derived from" substance X if it comprises a processed form of substance X, where processing can be one or more of: comminution, grinding, heat treatment, hydrolysis, and filtration. In this disclosure, the term "derived from" is used interchangeably and as a synonym with the terms "obtained from" or "derived from."

[0088] The collagen herein may be one or more selected from type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, and type X collagen.

[0089] Preferably, collagen hydrolysate as disclosed herein derives from one or more types of collagen, and the collagen is selected from type I collagen, type II collagen, type III collagen and type VI collagen. As a supplement or alternative, collagen hydrolysate as disclosed herein preferably derives from an animal raw material comprising different collagen subtypes (such as two or more of type I collagen, type II collagen, type III collagen and type IV collagen). As a supplement or alternative, collagen hydrolysate as disclosed herein can be a mixture of two or more of type I, type II, type III and type IV collagen.

[0090] The hydrolyzed collagen as disclosed herein is preferably derived from type I collagen, type II collagen, type III collagen and type IV collagen.

[0091] Collagen as disclosed herein may be derived from any one or more animals or animal species, such as bovine species, porcine species, chicken species, and fish species.

[0092] In one embodiment, the collagen is derived from cow. In one embodiment, the collagen as taught herein is derived from pig. In one embodiment, the collagen as taught herein is derived from fish. In one embodiment, the collagen as taught herein is derived from chicken.

[0093] In various embodiments, the collagen is a mixture of collagen from different sources (e.g., collagen derived from multiple animal species and / or collagen derived from different tissues). For example, the collagen as disclosed herein can be a mixture of two or more collagens selected from the group consisting of fish collagen, porcine collagen, chicken collagen, and bovine collagen.

[0094] In one embodiment, the collagen as disclosed herein is derived from an animal other than a bird, preferably an animal other than a chicken. In one embodiment, the chondroitin sulfate as disclosed herein is derived from tissue obtained from an animal other than a bird, preferably an animal other than a chicken. In one embodiment, the collagen hydrolyzate as disclosed herein is derived from tissue obtained from an animal other than a bird, preferably an animal other than a chicken. In one embodiment, the collagen-containing substance as disclosed herein does not contain tissue obtained from a bird, preferably does not contain tissue obtained from a chicken.

[0095] Collagen-containing materials as disclosed herein can be derived from one or more tissues selected from the group consisting of skin, scales, antlers, protrusions (e.g., humps), horns, head, brain, neck, ears, eyes, nose, tongue, lips, mouth, esophagus, trachea, sternum, larynx, bronchi, limbs, feet, toes, palms, claws, bones, cartilage, bone marrow, joints, membranes, hind legs, ligaments, tendons, ribs, diaphragms, muscles, skeletal muscle, smooth muscle, intestines, blood vessels, bladder, stomach, aorta, heart, liver, kidney, chest, lungs, spleen, pancreas, eggs, sperm, testicles, ovaries, nerves, gall bladder, and abdomen. As disclosed herein, the term "skin" encompasses "hide," meaning the outer covering of a large animal, such as from a cattle (species) or any other large animal. The terms "skin" and "hide" are used interchangeably herein and may refer to the outer covering of an animal, regardless of size.

[0096] In one embodiment, the collagen-containing material is derived from the trachea, sternum, or a mixture thereof.

[0097] In one embodiment, the collagen-containing substance comprises 20% to 80% by weight (e.g., 25% to 70% by weight, or 30% to 65% by weight, or 35% to 60% by weight, or 40% to 55% by weight) type I collagen and 20% to 80% by weight (e.g., 25% to 70% by weight, or 30% to 65% by weight, or 35% to 60% by weight, or 40% to 55% by weight) type II collagen, calculated based on the total weight of collagen in the collagen-containing substance.

[0098] In one embodiment, the collagen-containing substance comprises 1 wt% to 25 wt%, for example 2 wt% to 20 wt%, or 3 wt% to 15 wt%, or 4 wt% to 10 wt% collagen derived from type III collagen, calculated on the total weight of collagen in the collagen-containing substance.

[0099] In one embodiment, the collagen-containing material comprises 5% to 20% by weight of type III collagen, calculated on the total weight of collagen in the collagen-containing material.

[0100] In one embodiment, the collagen-containing material comprises 0.5 to 20 wt%, preferably 1 to 10 wt%, more preferably 2 to 5 wt% type IV collagen, calculated on the total weight of collagen in the collagen-containing material.

[0101] In a preferred embodiment, the present invention relates to a composition comprising 10% to 40% by weight of chondroitin sulfate and 60% to 90% by weight of collagen hydrolysate, calculated on the total weight of chondroitin sulfate and collagen hydrolysate.

[0102] The collagen hydrolysate preferably comprises 40% to 60% by weight of collagen derived from type I collagen, calculated based on the total weight of collagen in the collagen hydrolysate, and / or

[0103] The collagen hydrolysate preferably comprises 40% to 60% by weight of collagen derived from type II collagen, calculated based on the total weight of collagen in the collagen hydrolysate, and / or

[0104] The collagen hydrolysate preferably contains 5 wt % to 20 wt % of collagen derived from type III collagen, based on the total weight of collagen in the collagen hydrolysate.

[0105] The present invention also relates to a method for preparing the composition of the present invention by hydrolyzing a substance containing collagen, preferably comprising one or more of the following steps:

[0106] a) comminuting the collagen-containing material, preferably by grinding;

[0107] b) enzymatic hydrolysis;

[0108] c) a first separation step;

[0109] d) enzyme inactivation and heat treatment;

[0110] e) a second separation step;

[0111] f) concentrating the mixture;

[0112] g) drying.

[0113] In one embodiment, the enzymatic hydrolysis (step b) is carried out at a pH of 5 to 8, preferably a pH of 6 to 7. In one embodiment, the enzymatic hydrolysis (step b) is carried out at a temperature of 55 to 70° C., preferably 60 to 65° C. In one embodiment, the enzymatic hydrolysis (step b) is carried out for 4 to 7 hours, preferably 5 to 6 hours.

[0114] In one embodiment, the first separation step (step c) comprises subjecting the hydrolyzate (obtained in step c) to a temperature of 60 to 90° C., preferably 70 to 80° C. In one embodiment, the second separation step (step e) comprises subjecting the hydrolyzed collagen-containing material to a temperature of 75 to 99° C., preferably 85 to 99° C. In one embodiment, the second separation step (step e) comprises subjecting the hydrolyzed collagen-containing material to filtration.

[0115] The filtering herein (in step e) is preferably carried out by:

[0116] - using one or more water permeability of 2 to 2000 (l / min*m 2 at 1 bar), preferably 10 to 1000 (l / min*m 2 at 1 bar), more preferably 20 to 500 (l / min*m 2 filtration sheet at 1 bar); and / or

[0117] - using one or more filters with a separation degree of 0.02 to 100 (mK), preferably 0.1 to 50 (mK), more preferably 0.2 to 10 (mK); and / or

[0118] - using one or more materials having a weight per unit area of 100 to 6000 (g / m 2 ), preferably 500 to 4000 (g / m 2 ), more preferably 1000 to 2000 (g / m 2 ) filter.

[0119] One or more separation steps disclosed herein can determine the weight %, weight ratio or characteristics (such as molecular weight, dispersibility) of collagen hydrolysate, chondroitin sulfate and / or hyaluronic acid in the composition. As a supplement or alternative, one or more separation steps disclosed herein (i.e., step c and / or step e) can determine the sterility of the composition. In particular, it is found that the desired amount of chondroitin sulfate and / or hyaluronic acid in the composition can be achieved by one or more separation steps as disclosed herein.

[0120] In a preferred embodiment, the desired amount of hyaluronic acid in the composition, and / or the desired weight ratio of chondroitin sulfate to hyaluronic acid, is achieved by performing a second separation step (i.e., step e):

[0121] - at 65 to 99°C, preferably 75 to 99°C, more preferably 85 to 99°C; and / or

[0122] - using one or more water permeability of 2 to 2000 (l / min*m 2 at 1 bar), preferably 10 to 1000 (l / min*m 2 at 1 bar), more preferably 20 to 500 (l / min*m 2 filter discs at 1 bar); and / or

[0123] - using one or more filters with a separation degree of 0.02 to 100 (mK), preferably 0.1 to 50 (mK), more preferably 0.2 to 10 (mK); and / or

[0124] - using one or more materials having a weight per unit area of 100 to 6000 (g / m 2 ), preferably 500 to 4000 (g / m 2 ), more preferably 1000 to 2000 (g / m 2 ) filter.

[0125] Using one or more of the above steps in step e), a composition comprising a hyaluronic acid level lower than 0.5% by weight is obtained.

[0126] (Therapeutic) use of the composition

[0127] As part of the present invention, each of collagen hydrolysate and chondroitin sulfate or the combination thereof may independently act as an active ingredient in achieving a (therapeutic) effect (e.g. improving joint pain), e.g. this means that it induces a biological response in cells and / or the body.

[0128] In one embodiment, the compositions disclosed herein are used to improve pain, preferably joint pain. In one embodiment, the compositions disclosed herein are used to therapeutically improve pain, preferably therapeutically improve joint pain. In one embodiment, the compositions disclosed herein are used for non-therapeutic improvement of pain, preferably non-therapeutic improvement of joint pain.

[0129] In one embodiment, the compositions as disclosed herein are used to improve pain, preferably pain after exercise. In one embodiment, the compositions as disclosed herein are used to therapeutically improve pain, preferably pain after exercise. In one embodiment, the compositions as disclosed herein are used to non-therapeutic improve pain, preferably pain after exercise.

[0130] In one embodiment herein, the motion as disclosed herein is overload motion. In one embodiment, the motion as disclosed herein is strength training. In one embodiment, the motion as disclosed herein is endurance training. In one embodiment, the motion as disclosed herein is sports.

[0131] In one embodiment, the present invention is directed to a method of treating pain in a subject, preferably treating joint pain in a subject, more preferably treating joint pain after exercise in a subject, comprising administering to the subject a composition as disclosed herein.

[0132] According to the therapeutic use of the composition as disclosed herein, the method of treating pain may include one or more features described herein, including some embodiments of the composition, the formulation of the composition, the time of administering the composition, and the dosage of the composition for the therapeutic use of the composition.

[0133] In one embodiment, the present invention relates to the use of a composition as disclosed herein for the preparation of a medicament for the treatment of pain as disclosed herein.

[0134] The use of the composition for the preparation of a medicament as disclosed herein may include one or more of the features described herein for the therapeutic use of the composition as disclosed herein, including some embodiments of the composition, the formulation of the composition, the time of administering the composition, and the dosage of the composition for therapeutic use of the composition.

[0135] As used herein, "pain" (e.g., joint pain, pain after exercise) preferably means any level of pain that can be measured using a (validated) pain scale, preferably by one or more of the VAS, NPRS, and WOMAC scales. Additionally or alternatively, "pain" may be pain (e.g., joint pain, pain after exercise) determined by a professional medical practitioner.

[0136] The visual analogue scale (VAS) score is a measure of acute and chronic pain. In the case of the VAS, the score is recorded by the subject making a handwritten mark on a 100 mm line, which represents a continuum between "no pain" and "severe pain" (Breivik et al. Br. J. Anaesth. 2008 Jul; 101 (1): 17-24). In one embodiment, "pain" is defined by a VAS score of at least 1 mm, preferably at least 10 mm, more preferably at least 40 mm, and even more preferably at least 70 mm. In one embodiment, when a subject reports pain specifically experienced in one or more joints, "joint pain" is defined by a VAS score of at least 1 mm, preferably at least 10 mm, more preferably at least 40 mm, and even more preferably at least 70 mm. In one embodiment, when a subject reports pain specifically experienced in one or more joints after exercise (e.g., within 24 hours, 48 hours, or 72 hours after exercise), "joint pain after exercise" is defined by a VAS score of at least 1 mm, preferably at least 10 mm, more preferably at least 40 mm, and even more preferably at least 70 mm.

[0137] In one embodiment, "improvement" of pain as used herein means a decrease in the VAS score of at least 1 mm, preferably at least 10 mm, more preferably at least 20 mm (e.g., when the VAS score is compared to a control that did not receive the composition of the invention, and / or when the VAS score before treatment with the composition of the invention is compared to the VAS score after treatment with the composition of the invention).

[0138] Numeric Pain Rating Scale (NPRS) is a measure of acute pain and chronic pain, in which subjects rate their pain on an 11-point numerical scale. The scale is composed of 0 (no pain at all) to 10 (imaginable severe pain) (Hwaker et al. Arthritis Care Res. (Hoboken). 2011 Nov; 63 Suppl. 11: S240-52). In one embodiment, "pain" is defined by at least 1, preferably at least 3, more preferably at least 5, and even more preferably at least 7 NPRS scores. In one embodiment, when a subject reports pain specifically experienced in one or more joints, "joint pain" is defined by at least 1, preferably at least 3, more preferably at least 5, and even more preferably at least 7 NPRS scores. In one embodiment, when a subject reports pain specifically experienced in one or more joints after exercise (e.g., within 24 hours, 48 hours, or 72 hours after exercise), "joint pain after exercise" is defined by at least 1, preferably at least 3, more preferably at least 5, and even more preferably at least 7 NPRS scores.

[0139] In one embodiment, "improvement" of pain herein means a decrease in the NPRS score of at least 1, preferably at least 2, more preferably at least 3 (e.g., when the NPRS score is compared to a control that did not receive the composition of the invention, and / or when the NPRS score before treatment with the composition of the invention is compared to the NPRS score after treatment with the composition of the invention).

[0140] The Western Ontario and McMaster Universities osteoarthritis index (WOMAC) score is a measure of osteoarthritis severity with subscale scores for pain, stiffness, and joint physical function (Bellamy et al. J. Rheumatol. 1988 Dec; 15(12): 1833-40). The WOMAC measures five items for pain (scoring range 0 to 20), two items for stiffness (scoring range 0 to 8), and 17 items for functional limitation (scoring range 0 to 68). Items are rated using one of five possible responses (0=none, 1=mild, 2=moderate, 3=severe, 4=extreme).

[0141] In one embodiment, "pain" is defined by a WOMAC score (rated using one of five responses: 0 = none, 1 = mild, 2 = moderate, 3 = severe, 4 = extreme) of one or more pain items of at least 1, preferably at least 2, more preferably at least 3, even more preferably 4. In one embodiment, when a subject reports pain specifically experienced in one or more joints, "joint pain" is defined by a WOMAC score of one or more pain items of at least 1, preferably at least 2, more preferably at least 3, even more preferably 4. In one embodiment, when a subject reports pain specifically experienced in one or more joints after exercise (e.g., within 24 hours, 48 hours, or 72 hours after exercise), "joint pain after exercise" is defined by a WOMAC score of one or more pain items of at least 1, preferably at least 2, more preferably at least 3, even more preferably 4.

[0142] In one embodiment, "improvement" of pain as used herein means a decrease in the WOMAC score (rated using one of five responses: 0 = none, 1 = mild, 2 = moderate, 3 = severe, 4 = extreme) for one or more pain items by at least 1, preferably at least 2, more preferably at least 3 (e.g., when the WOMAC score is compared to a control that did not receive the composition of the invention, and / or when the WOMAC score before treatment with the composition of the invention is compared to the WOMAC score after treatment with the composition of the invention).

[0143] The present inventors have found that the use of the composition of the present invention has positive results in alleviating joint pain regardless of gender and exercise intensity.

[0144] The present inventors also found positive results in alleviating joint pain in all age categories, with the greatest reduction in joint pain across age categories being observed in relatively young subjects (e.g., <30 years old). Since increasing age is a risk factor for joint diseases (primarily osteoarthritis), the compositions of the present invention may be particularly effective in subjects who do not suffer from joint diseases (primarily osteoarthritis).

[0145] In one embodiment, the composition as disclosed herein is used to alleviate pain in a subject in the age category < 30 years.

[0146] In one embodiment, the compositions as disclosed herein are used to relieve pain in male and / or female subjects.

[0147] In one embodiment, the compositions as disclosed herein are used to reduce pain from moderate exercise and / or low joint impact activities such as walking, swimming, flexibility / balance, gardening, cycling, dancing, and horseback riding.

[0148] In one embodiment, a composition as disclosed herein is used to reduce pain from moderate intensity activities and / or moderate joint impact activities, such as weight lifting, gym cardio, or boxing.

[0149] In one embodiment, the composition as disclosed herein is used to reduce pain from high intensity activities and / or high joint impact activities, such as running, ball games, rope skipping or (heavy) weight lifting.

[0150] Therapeutic and non-therapeutic uses

[0151] The compositions disclosed herein may have both non-therapeutic and therapeutic uses in ameliorating pain. Non-therapeutic or therapeutic uses may be distinguished based on the nature of the pain and / or different groups of subjects experiencing pain.

[0152] The first group (herein " healthy subject group ") includes healthy individuals who do not obtain therapeutic benefits from the treatment using the compositions of the present invention. For example, pain can be non-pathological, which means that pain generally has a severity that is not expected to cause health problems or (serious) suffering. In addition or as an alternative, pain can have a cause or severity that causes the pain to disappear naturally over time (i.e., it is not chronic). In addition or as an alternative, the pain severity in the healthy subject group is limited so that they generally do not seek help from professional medical practitioners. For example, when pain mainly occurs after exercise and pain generally disappears in the absence of additional intervention, healthy subjects generally do not seek help from medical practitioners. In this example, non-therapeutic interventions can still achieve faster pain relief. Compared with objects in which pain has pathological properties, the healthy subject group may need less or shorter treatment using the compositions of the present invention to prevent and / or treat pain.

[0153] The second group (herein referred to as the "pathological subject group") includes subjects whose pain is of a pathological nature, meaning that the pain causes severe pain symptoms and (severe) distress and / or may lead to (serious) health and psychological risks. In addition, in the pathological subject group, the pain may be chronic. The severity of the pain is such that help from a professional medical practitioner is often sought. Compared to subjects whose pain is of a non-pathological nature, the pathological subject group may require more or longer treatment with the composition of the present invention.

[0154] A professional medical practitioner is generally able to determine on a case-by-case basis whether a pain condition requires therapeutic intervention or non-therapeutic intervention (i.e., whether the individual falls into the group of healthy subjects or pathological subjects). For example, a professional medical practitioner can determine whether the pain may lead to (serious) health and psychological risks and / or whether the pain is chronic.

[0155] In one embodiment, the present invention relates to the use of a composition for non-therapeutic improvement of pain. In a preferred embodiment, the present invention relates to the use of a composition for non-therapeutic improvement of joint pain. In a preferred embodiment, the present invention relates to the use of a composition for non-therapeutic improvement of pain after exercise. In a preferred embodiment, the present invention relates to the use of a composition for non-therapeutic improvement of joint pain after exercise.

[0156] In one embodiment, the present invention relates to a composition for (therapeutic) improving pain, wherein the pain is preferably one or more selected from the group consisting of fibromyalgia, chronic low back pain, headache, migraine, tension headache, irritable bowel syndrome, chronic pelvic pain, phantom limb pain, carpal tunnel syndrome, sciatica, postherpetic neuralgia, gout, cancer pain, neuropathic pain, complex regional pain syndrome (CRPS), myofascial pain syndrome and endometriosis-associated pain.

[0157] In one embodiment, the present invention relates to a composition for the (therapeutic) improvement of pain in joint diseases.

[0158] In a preferred embodiment, the joint disease is one or more selected from the group consisting of osteoarthritis, degenerative joint diseases, rheumatoid arthritis, polymyalgia rheumatica, juvenile arthritis, gout, ankylosing spondylitis, psoriatic arthritis, chondromalacia patellae, periarticular diseases and costochondritis.

[0159] In one embodiment, the composition as disclosed herein is used to relieve pain in a subject not suffering from a joint disease, wherein a joint disease preferably means one or more of osteoarthritis, rheumatoid arthritis, septic arthritis, juvenile idiopathic arthritis, lupus, gout and bursitis.

[0160] Administration of the composition

[0161] The term "composition" as used herein preferably means that the individual components (e.g., collagen hydrolyzate, chondroitin sulfate) are provided as a physical combination (i.e., in a single formulation). The term "composition" as used herein does not exclude that the individual components (e.g., collagen hydrolyzate, chondroitin sulfate) are not in the form of a physical combination, e.g., in the form of a separate formulation as part of a combination or kit of parts. The optimal administration regimen of collagen hydrolyzate and chondroitin sulfate can be determined based on the specific circumstances and, for example, depends on the subject to be treated, the condition to be treated, or the formulation used.

[0162] In one embodiment, the individual components (e.g., collagen hydrolysate, chondroitin sulfate) are in the form of separate preparations, e.g., as part of a combination. In one embodiment, the individual components (e.g., collagen hydrolysate, chondroitin sulfate) are in the form of a single preparation (i.e., composition). In one embodiment, the individual components (e.g., collagen hydrolysate, chondroitin sulfate) are administered separately, sequentially, or simultaneously with each other, e.g., as part of a combination. In one embodiment, the individual components (e.g., collagen hydrolysate, chondroitin sulfate) are packaged separately in the form of a "kit of parts."

[0163] When the individual components (e.g., collagen hydrolysate, chondroitin sulfate) are administered not too far apart from each other, the effect of the composition may be more pronounced, for example, for improving the biological activity of (the interaction between) collagen hydrolysate and chondroitin sulfate. In one embodiment, the administration of collagen hydrolysate and chondroitin sulfate is no more than 24 hours apart, preferably no more than 12 hours apart, more preferably no more than 6 hours apart, even more preferably no more than 2 hours apart (e.g., 60, 30 or 15 minutes).

[0164] In a preferred embodiment, the collagen hydrolysate and / or chondroitin sulfate as disclosed herein is administered orally, wherein the collagen hydrolysate and chondroitin sulfate are in the form of a single formulation (ie, a composition) or in the form of separate formulations.

[0165] In one embodiment, the collagen hydrolysate and / or chondroitin sulfate as disclosed herein is provided in the form of one or more formulations selected from the group consisting of a feed formulation, a feed supplement formulation, or a pharmaceutical formulation, wherein the collagen hydrolysate and chondroitin sulfate are provided in the form of a single formulation (i.e., a composition) or in the form of separate formulations.

[0166] In one embodiment, the collagen hydrolysate and / or chondroitin sulfate as disclosed herein is provided in the form of one or more formulations selected from the group consisting of capsules, tablets, or powders, preferably powders, wherein the collagen hydrolysate and chondroitin sulfate are provided in the form of a single formulation (i.e., a composition) or in the form of separate formulations.

[0167] In one embodiment, the collagen hydrolysate and / or chondroitin sulfate as disclosed herein is provided in the form of one or more formulations selected from the group consisting of a drinkable solution or suspension, a drink such as a syrup, a (water-soluble) powdered mixture, a (water-soluble) paste, a (water-soluble) powder, a (water-soluble) tablet, a (water-soluble) pill, a (water-soluble) dragee, a (water-soluble) caplet, a (water-soluble) sachet or a (water-soluble) capsule.

[0168] In one embodiment, the composition as disclosed herein (for use) is administered in a daily dose of 20 mg to 50 g, preferably 50 mg to 20 g, more preferably 0.1 g to 10 g, even more preferably 0.2 g to 2 g, wherein the daily dose is the total dry weight of the composition.

[0169] In one embodiment, the composition as disclosed herein (for use) is administered in a unit dose of 10 mg to 25 g, preferably 25 mg to 10 g, more preferably 50 mg to 5 g, even more preferably 0.1 g to 1 g, wherein the unit dose is the total dry weight of the composition.

[0170] The daily dose and / or unit dose of the composition as disclosed herein (for use) can be at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50, all in g, wherein the dose is the dry weight of the composition administered to the subject. Additionally or alternatively, in various embodiments, the daily dose and / or unit dose of the composition as disclosed herein (for use) does not exceed 50, 45 40, 35, 30, 25, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 or 0.1, all in g, wherein the dose is the dry weight of the composition administered to the subject.

[0171] As used herein, the term "daily dose" refers to the total dry weight administered to a subject daily.

[0172] The daily dose of the composition can be administered as a single unit dose, or as two, three, four or more unit doses. The amounts of the two or more unit doses may be the same or different. The daily dose of the composition as disclosed herein is preferably administered as two unit doses, more preferably as two unit doses each corresponding to 30% to 70%, preferably 40% to 60% of the amount of the daily dose.

[0173] In a preferred embodiment, the dosage regimen for the composition as disclosed herein comprises administering the daily dose of the composition as two unit doses, wherein:

[0174] - an amount per unit dose of 0.1 to 2 g, preferably 0.2 to 1 g, more preferably 0.3 to 0.7 g, wherein the amount is by dry weight of the composition; and / or

[0175] - The two unit doses are administered at least 6 hours, preferably 8 hours, more preferably 12 hours apart from each other.

[0176] The term "unit dose" as used herein relates to the total dry weight administered to a subject in a single dose. A unit dose is typically a pre-prepared form (e.g., a pre-packaged dose) ready for administration. For example, a unit dose can also be identified from the product packaging or label.

[0177] "Daily dose" and "unit dose" refer to the total dry weight, and also refer to the total dry weight when the individual components (e.g., collagen hydrolysate, chondroitin sulfate) are administered as a combination of separate formulations. For example, when 2 g of collagen hydrolysate from a first formulation and 2 g of chondroitin sulfate from a second formulation are administered to a subject, 4 g of the composition is considered to have been administered to the subject. For example, when 0.3 g of collagen hydrolysate from a first formulation and 0.2 g of chondroitin sulfate from a second formulation are administered to a subject, 0.5 g of the composition is considered to have been administered to the subject.

[0178] The present inventors observed the greatest reduction in joint pain in healthy subjects starting three weeks after starting daily administration (2×0.5 g / day) of the composition. The reduction in joint pain was maintained by daily administration (2×0.5 g / day) of the composition. After stopping administration of the composition (i.e., during the "washout period"), the reduction in joint pain was observed to last for at least another 4 weeks (e.g., 6 to 8 weeks). This suggests that, particularly in healthy subjects after exercise, the composition is preferably administered daily, and preferably over a long period of time (e.g., three or more consecutive weeks).

[0179] In one embodiment, a composition as disclosed herein is administered to a subject repeatedly, preferably at least once daily or at least once every other day.

[0180] In one embodiment, a composition as disclosed is administered to a subject for at least two consecutive weeks, preferably at least four consecutive weeks, more preferably at least six consecutive weeks, and most preferably at least eight consecutive weeks.

[0181] In one embodiment, the composition is administered for at least 2, 3, 4, 5, 6, 7, 10, 14, or 21 days, preferably consecutive days, before improvement in pain is achieved or is most effective.

[0182] In one embodiment, daily administration of the composition for at least three weeks results in joint pain reduction for at least an additional 4 weeks, preferably at least an additional 6 weeks, more preferably at least an additional 8 weeks after cessation of administration of the composition (ie, during a washout period).

[0183] definition

[0184] - The term "enzymatic hydrolysis" as used herein means the process of enzymatically cleaving one or more bonds in collagen using one or more enzymes, preferably resulting in the formation of smaller (peptide) fragments. "Enzymatic hydrolysis" herein encompasses partial hydrolysis.

[0185] As used herein, the term "amelioration" encompasses both "prevention" and "treatment" of a condition. The term "prevention" means ensuring that a subject does not develop a condition (e.g., pain, joint pain). Intervention is considered a form of "prevention" herein when the condition is delayed, reduced in severity, and / or reduced in incidence, even when the condition is not completely prevented from occurring. As used herein, "prevention" (or variations thereof) by intervention encompasses situations in which a subject has previously experienced a condition (e.g., pain, joint pain) but the intervention prevents the condition from recurring. "Prevention" (or variations thereof) can have therapeutic and / or non-therapeutic effects. If "prevention" (or variations thereof) is therapeutic in nature, it can also target the symptoms of the disease or condition and / or its underlying pathological condition. "Prevention" (or variations thereof) can be defined by any delay, change in severity, and / or change in incidence, for example, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% compared to a control or reference, as measured by any standard technique. As used herein, "treating" means that once a condition already exists, an intervention is performed to alleviate and / or cure a condition (e.g., pain, joint pain). "Treatment" can have therapeutic and / or non-therapeutic effects. If "treatment" is therapeutic in nature, it can be directed to the symptoms of a disease or condition and / or its underlying pathological conditions. "Treatment" can be any reduction in the severity, incidence, and / or frequency of, for example, a condition, such as at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% compared to a control or reference, as measured by any standard technique. "Improvement" as used herein also encompasses "curing." The terms "improve" and "reduce" as used herein can be used interchangeably with "reduce" or "reduce."

[0186] As used herein, the terms "comprises / comprising" or "to comprise / including" and variations thereof refer to instances where the terms are used in their non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. They also encompass the more restrictive verbs "consisting essentially of" and "consisting of."

[0187] Unless the context clearly requires the presence of one and only one element, the mention of an element by a noun without a quantifier does not exclude the possibility that more than one element is present. Thus, a noun without a quantifier generally means "at least one".

[0188] As used herein, a level is "increased" or "decreased" when the level is at least 1% higher or lower (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%) than the corresponding level in a control or reference, respectively. Alternatively, the level in a sample is considered increased or decreased when the level in the sample is statistically significantly higher or lower, respectively, as compared to the level in the control or reference, including at an earlier time point. The terms "for / to reduce" and "for / to decrease" are used interchangeably herein. The terms "decreased" and "decreased" are used interchangeably herein.

[0189] - As used herein, the term "subject" or "individual" refers to any animal (eg, a mammal), preferably a human.

[0190] As used herein, the term "administering" or variations thereof includes providing a compound, substance, or composition to a subject who consumes the compound, substance, or composition. A subject who consumes the compound, substance, or composition may administer the compound, substance, or composition to himself / herself. In such cases, the term "administering" may be considered "taking in."

[0191] As used herein, the term "unit dose" refers to an amount or unit of, for example, a compound, substance, or composition, administered to / ingested by a subject as a single dose. For example, a unit dose can be a pre-prepared form (e.g., a prepackaged dose) ready for administration to a subject. For example, a unit dose can be identifiable from the product packaging or label. A total daily dose can be divided into multiple unit doses, each having a reduced dose compared to the total daily dose.

[0192] The term "joint" as used herein preferably refers to the group consisting of: knee, shoulder, hip, finger joint, elbow, toe joint, neck joint, wrist joint, facet joint or more thereof. In one embodiment, the joint is a knee joint. In one embodiment, the joint is a shoulder joint. In one embodiment, the joint herein is a hip joint.

[0193] As used herein, the term "exercise" refers to any physical activity that can be planned, organized, and has an ultimate or intermediate goal. As used herein, the term "exercise" generally has the goal of improving or maintaining physical fitness. The terms "exercise" and "physical movement" are used interchangeably herein. For example, "exercise" may include all professional or non-professional activities, including: sports, strength or resistance training, and endurance or aerobic activities. In addition or alternatively, "exercise" may include endurance training. The term "endurance training" refers to training the aerobic system as opposed to the anaerobic system, for example, by performing physical exercise at an elevated heart rate for a prolonged period of time (e.g., at 70% of maximum heart rate (MHR) for at least 30 minutes) during walking, running, jogging, cycling, swimming, jumping rope, etc. In the present disclosure, the term "exercise" encompasses "exercise overload." As used herein, the term "exercise overload" refers to physical overload caused by high strain and / or force applied to a body part (e.g., a joint) during any form of physical exercise. Overload is typically caused by acute overload, but it can also be caused by repeated overload. Exercise overload, most importantly acute overload, often results in high strain on joints (eg, knees, shoulders, hips), which can lead to microdamage.

[0194] - In the context of administration herein, the term "continuous" or "continuously" as used herein means administration one after another in sequence without a break within a given time period. For example, when "the composition is administered for 4 consecutive days", this means that the composition is administered at least once a day for 4 consecutive days (e.g., Monday, Tuesday, Wednesday, and Thursday of the same week), regardless of the number of administrations per day or the total number of administrations. For example, when "the composition is administered for 4 consecutive weeks", this means that the composition is administered at least once a week for 4 consecutive weeks, regardless of the number of administrations per day, the number of administrations per week, or the total number of administrations.

[0195] Terms

[0196] In this document, the clauses are some embodiments of the present invention. The features of the clauses (embodiments) in this document can be combined.

[0197] Clause 1:

[0198] A composition comprising 10% to 40% by weight of chondroitin sulfate and 60% to 90% by weight of collagen hydrolysate, calculated based on the total weight of the chondroitin sulfate and the collagen hydrolysate,

[0199] The collagen hydrolysate can be obtained by hydrolyzing a substance containing collagen,

[0200] The collagen-containing substance comprises 40% to 60% by weight of type I collagen and 40% to 60% by weight of type II collagen, calculated based on the total weight of collagen in the collagen-containing substance.

[0201] Clause 2:

[0202] The composition according to item 1, further comprising 5% to 20% by weight of type III collagen, calculated based on the total weight of collagen in the collagen-containing material.

[0203] Clause 3:

[0204] The composition according to clause 1 or 2, wherein the average molecular weight of the collagen hydrolysate is greater than 1000 Da and less than 10000 Da.

[0205] Clause 4:

[0206] The composition according to clause 3, wherein the average molecular weight of the collagen hydrolysate is greater than 1000 Da and less than 5000 Da.

[0207] Article 5:

[0208] A composition according to any of the preceding clauses, wherein the collagen hydrolysate comprises 2 to 20 wt% hydroxyproline and / or 0.1 to 5 wt% hydroxylysine, wherein the wt% is calculated based on the total weight of amino acids in the collagen hydrolysate.

[0209] Article 6:

[0210] A composition according to any of the preceding clauses, wherein the collagen hydrolysate comprises hydroxyproline and hydroxylysine in a molar ratio (hydroxyproline:hydroxylysine) greater than 8 and less than 15.

[0211] Article 7:

[0212] The composition according to any of the preceding clauses, comprising 15 to 40 wt%, preferably 20 to 40 wt%, more preferably 30 to 40 wt% chondroitin sulfate, calculated on the total weight of the chondroitin sulfate and the collagen hydrolysate.

[0213] Article 8:

[0214] A composition according to any of the preceding clauses, wherein the chondroitin sulfate is obtained from the hydrolysis of the collagen-containing material.

[0215] Article 9:

[0216] A composition according to any of the preceding clauses, wherein the hydrolysis is enzymatic hydrolysis.

[0217] Article 10:

[0218] A composition according to any of the preceding clauses, wherein the collagen-containing material is derived from one or more cartilage-containing tissues.

[0219] Article 11:

[0220] The composition according to clause 10, wherein the tissue is the trachea and / or sternum.

[0221] Article 12:

[0222] A composition comprising 10% to 40% by weight of chondroitin sulfate and 60% to 90% by weight of collagen hydrolysate, calculated based on the total weight of the chondroitin sulfate and the collagen hydrolysate,

[0223] wherein the collagen hydrolysate comprises 40% to 60% by weight of collagen derived from type I collagen, calculated based on the total weight of collagen in the collagen hydrolysate; and

[0224] The collagen hydrolysate comprises 40 wt % to 60 wt % of collagen derived from type II collagen, calculated based on the total weight of collagen in the collagen hydrolysate.

[0225] Article 13:

[0226] The composition according to clause 12, wherein the collagen hydrolysate further comprises 5% to 20% by weight of collagen derived from type III collagen, calculated on the total weight of collagen in the collagen hydrolysate.

[0227] Article 14:

[0228] A composition according to clause 12 or 13, wherein the average molecular weight of the collagen hydrolysate is greater than 1000 Da and less than 10000 Da.

[0229] Article 15:

[0230] The composition according to clause 14, wherein the average molecular weight of the collagen hydrolysate is greater than 1000 Da and less than 5000 Da.

[0231] Article 16:

[0232] A composition according to any one of clauses 12 to 15, wherein the collagen hydrolysate comprises 2 to 20 wt% hydroxyproline and / or 0.1 to 5 wt% hydroxylysine, wherein the wt% is calculated based on the total weight of amino acids in the collagen hydrolysate.

[0233] Article 17:

[0234] A composition according to any one of clauses 12 to 16, wherein the collagen hydrolysate comprises hydroxyproline and hydroxylysine in a molar ratio (hydroxyproline:hydroxylysine) greater than 8 and less than 15.

[0235] Article 18:

[0236] A composition according to any one of items 12 to 17, comprising 15 to 40 wt%, preferably 20 to 40 wt%, more preferably 30 to 40 wt% chondroitin sulfate, calculated on the total weight of the chondroitin sulfate and the collagen hydrolysate.

[0237] Article 19:

[0238] A composition according to any one of clauses 1 to 18 for use in therapeutic treatment.

[0239] Article 20:

[0240] A composition according to any one of clauses 1 to 18, for use in the therapeutic improvement of pain.

[0241] Article 21:

[0242] A composition according to any one of clauses 1 to 18, for use in the non-therapeutic improvement of pain.

[0243] Article 22:

[0244] A composition for use according to clause 20 or 21, wherein the pain is joint pain.

[0245] Article 23:

[0246] A composition for use according to clause 22, wherein the joint pain is after exercise.

[0247] Article 24:

[0248] A composition for use according to any one of clauses 19 to 23, wherein the composition is administered orally.

[0249] Article 26:

[0250] A composition for use according to any one of clauses 19 to 24, wherein the composition is administered in a daily dose of 0.2 to 2 g, wherein the daily dose is the total dry weight of the composition.

[0251] Article 27:

[0252] Use of the composition in non-therapeutic pain relief,

[0253] The composition comprises 10% to 40% by weight of chondroitin sulfate and 60% to 90% by weight of collagen hydrolysate, calculated based on the total weight of the chondroitin sulfate and the collagen hydrolysate.

[0254] The collagen hydrolysate contains hydroxyproline and hydroxylysine, and the molar ratio (hydroxyproline:hydroxylysine) thereof is greater than 8 and less than 15.

[0255] Clause 28: The use according to clause 27, wherein the pain is joint pain.

[0256] Article 29:

[0257] 28. The use according to clause 27 or 28, wherein the pain is after exercise.

[0258] Article 30:

[0259] 29. The use according to any one of clauses 27 to 29, wherein the collagen hydrolysate comprises from 2 wt% to 20 wt% hydroxyproline, wherein the wt% is calculated based on the total weight of amino acids in the collagen hydrolysate.

[0260] Article 31:

[0261] 3. The use according to any one of clauses 27 to 30, wherein the collagen hydrolysate comprises from 0.1 wt% to 5 wt% hydroxylysine, wherein the wt% is calculated based on the total weight of amino acids in the collagen hydrolysate.

[0262] Article 32:

[0263] The use according to any one of clauses 27 to 31, wherein the composition comprises 15% to 40% by weight, preferably 20% to 40% by weight, more preferably 30% to 40% by weight, of chondroitin sulfate, calculated based on the total weight of the chondroitin sulfate and the collagen hydrolysate.

[0264] Article 33:

[0265] Use according to any one of clauses 27 to 32, wherein the average molecular weight of the collagen hydrolysate is greater than 1000 Da and less than 10000 Da.

[0266] Article 34:

[0267] The use according to any one of clauses 27 to 33, wherein the composition is administered orally.

[0268] Article 35:

[0269] 3. The use according to any one of clauses 27 to 34, wherein the composition is administered in a daily dose of 0.2 to 2 g, wherein the daily dose is the total dry weight of the composition.

[0270] Article 36:

[0271] Composition for improving pain in joint diseases,

[0272] The composition comprises 10% to 40% by weight of chondroitin sulfate and 60% to 90% by weight of collagen hydrolysate, calculated based on the total weight of the chondroitin sulfate and the collagen hydrolysate.

[0273] The collagen hydrolysate contains hydroxyproline and hydroxylysine, and the molar ratio (hydroxyproline:hydroxylysine) thereof is greater than 8 and less than 15.

[0274] Article 37:

[0275] The composition for use according to clause 36, wherein the joint disease is one or more selected from the group consisting of osteoarthritis, degenerative joint diseases, rheumatoid arthritis, polymyalgia rheumatica, juvenile arthritis, gout, ankylosing spondylitis, psoriatic arthritis, chondromalacia patellar, periarticular diseases and costochondritis.

[0276] Article 38:

[0277] A composition for use according to any one of clauses 36 to 37, wherein the collagen hydrolysate comprises 2 wt% to 20 wt% hydroxyproline, wherein the wt% is calculated based on the total weight of amino acids in the collagen hydrolysate.

[0278] Article 39:

[0279] A composition for use according to any one of clauses 36 to 38, wherein the collagen hydrolysate comprises from 0.1 wt% to 5 wt% hydroxylysine, wherein the wt% is calculated based on the total weight of amino acids in the collagen hydrolysate.

[0280] Article 40:

[0281] The composition for use according to any one of clauses 36 to 39, comprising 15% to 40% by weight, preferably 20% to 40% by weight, more preferably 30% to 40% by weight, of chondroitin sulfate, calculated based on the total weight of the chondroitin sulfate and the collagen hydrolysate.

[0282] Article 41:

[0283] A composition for use according to any one of clauses 36 to 40, wherein the average molecular weight of the collagen hydrolysate is greater than 1000 Da and less than 10000 Da.

[0284] Article 42:

[0285] A composition for use according to any one of clauses 36 to 41 , wherein the composition is administered orally.

[0286] Article 44:

[0287] A composition for use according to any one of clauses 36 to 42, wherein the composition is administered in a daily dose of 0.2 to 2 g, wherein the daily dose is the total dry weight of the composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0288] Figure 1 Effect of a collagen hydrolysate-based composition (HC) compared to placebo on joint pain perception over a 16-week period. Supplement intake was stopped after 12 weeks (indicated by the vertical dashed line). The numbers in the figure correspond to the number of subjects.

[0289] Figure 2 Effect of a collagen hydrolysate-based composition (HC) compared to placebo on joint pain by sex. The study period was 16 weeks. Supplement intake was discontinued after 12 weeks (indicated by the vertical dashed line). The number of participants in the entire study is indicated in the figure.

[0290] Figure 3 Effect of a collagen hydrolysate-based composition (HC) compared to placebo on joint pain by age category. The study period was 16 weeks. Supplement intake was discontinued after 12 weeks (indicated by the vertical dashed line). The number of participants in the entire study is indicated in the figure.

[0291] Figure 4 Effect of a collagen hydrolysate-based composition (HC) compared to placebo on joint pain (according to physical activity). The study period was 16 weeks. Supplement intake was discontinued after 12 weeks (indicated by the vertical dashed line). The number of participants in the entire study is indicated in the figure.

[0292] Example

[0293] Example 1

[0294] The following protocol is a general protocol for preparing a composition comprising collagen hydrolysate and chondroitin sulfate, starting with a mixture comprising porcine tracheal and sternal tissue.

[0295] The amount of collagen types I, II, and III in the tissues was determined by ELISA. The relative amounts of tracheal and sternal tissues were combined to achieve a weight ratio of collagen types I, II, and III in the starting mixture of approximately 5:5:1.

[0296] The general approach includes the following steps:

[0297] a) grinding a substance containing collagen;

[0298] b) enzymatic hydrolysis (pH = 6 to 7, T = 60 to 65° C., t = 5 to 6 hours);

[0299] c) a first separation step and heat treatment (T = 70 to 80° C.);

[0300] d) enzyme activation and heat treatment (T>90°C, t>15 minutes);

[0301] e) A second separation step using a water permeability of 20 to 500 (l / min*m 2 at 1 bar), a separation degree of 0.2 to 10 (mK), a weight per unit area of 1000 to 2000 (g / m 2 ) and heat treatment (T = 85 to 99 ° C)

[0302] f) concentrating under vacuum;

[0303] g) drying.

[0304] The above protocol results in a composition comprising collagen hydrolysate and chondroitin sulfate having the general characteristics according to Table 1. In Examples 2 to 4, this product is referred to as "HC".

[0305] Variations in starting materials and protocols can be employed to modulate composition and biological activity (as shown in Examples 3 and 4).

[0306] Table 1. Product characteristics and nutritional information of compositions based on collagen hydrolysate and chondroitin sulfate (also referred to as "HC" in the examples). AA = amino acid.

[0307]

[0308]

[0309] Example 2

[0310] The purpose of this study was to understand how the average consumer experiences a collagen hydrolysate-based supplement in improving joint pain. A supplement was prepared from porcine trachea and sternum according to Example 1. The supplement, referred to herein as "HC," was provided by Rousselot BV (Ghent, Belgium).

[0311] Materials and methods

[0312] Study population and design

[0313] This consumer lifestyle study was a placebo-controlled (ratio 5:1, 177 participants using HC and 36 participants using placebo), blinded, randomized, parallel trial to evaluate the effects of collagen hydrolysate compared with placebo on joint pain.

[0314] To represent the general population of consumers who might seek nutritional supplements to relieve joint pain, the study aimed to collect data from a broad range of demographically diverse individuals. Participants were recruited through multiple sports and health clubs across the United Kingdom. To obtain relevant and relevant data, the researchers aimed to collect data from at least 200 participants.

[0315] Using a placebo-controlled design, participants took either 2 x 0.5g of HC (experimental group) or 2 x 0.5g of maltodextrin (placebo group) daily for 12 weeks and a 4-week run-in period (without a run-out period). Participants completed a self-assessment tool for all 16 weeks. HC and placebo were matched in taste, color, and aroma. Both used similar packaging.

[0316] All communication between researchers and participants was done through an online application (app), where they had to fill out their self-assessments and other observations.

[0317] Inclusion criteria for participants in the study were "18 years old," "no medical diagnosis," "not taking any medications," "regularly physically active," and "not pregnant or currently breastfeeding." For physical activity, this meant everything from gardening and brisk walking to marathon training.

[0318] Exclusion criteria were limited to “age below 18 years old”, “currently receiving medical diagnosis”, “currently taking / prescribed medication”, “not recommended or not regularly participating in physical activity”, and “currently pregnant or breastfeeding”.

[0319] Participants were also excluded if they were already using any type of supplement containing the following: vitamin D, curcumin / turmeric, other collagen, green mussel extract, boswellia, glucosamine, chondroitin sulfate, hyaluronic acid, or folic acid.

[0320] Participants who had supplemented with multivitamins, iron, omega 3, vitamin B complex, vitamin C, calcium, rosehip extract, amino acid complex, or creatine were not excluded, as these supplements are not specific for joint health and the results could potentially show any relationship with HC.

[0321] Material

[0322] The product used in this study is derived from hydrolyzed porcine trachea and sternum. The starting material is hydrolyzed (according to Example 1) to produce a composition with collagen hydrolyzate and chondroitin sulfate (hereinafter referred to as "HC"). The typical product characteristics of this product are according to Example 1. The HC dosage form is a 0.5g hydroxypropylmethylcellulose (HPMC) capsule, and the 0.5g maltodextrin in the HPMC capsule is used as a placebo. Maltodextrin is a food grade substance and is typically used as a placebo in clinical trials. This product is a tasteless and odorless white powder, does not contain micronutrient value, and therefore can be used as a placebo in this experiment.

[0323] Questionnaire design

[0324] In order to have continuous follow-up of participants, a smartphone application was developed for participants to submit their feedback on a weekly basis.

[0325] The weekly report consists of several questions. First, they are asked which physical activity they have recently completed and the duration of the activity. Preset activities (such as gardening, brisk walking, running, cycling, tennis, swimming, strengthening exercise (Strengthening sport), weightlifting (average weight squat 70kg), flexibility / balance, yoga, Pilates (Pilates)) or the option box to fill in any unlisted activity are provided. In terms of duration, participants are asked to check the overall amount each week and then indicate the amount of time spent (0 to 5 hours, 6 to 10 hours, 11 to 15 hours, 16 to 20 hours or 20 hours or longer).

[0326] Next, they received a body outline. Here, they could indicate the area where they experienced joint pain. Then, participants were asked to give their feedback on their joint pain using a validated pain measurement instrument, the Visual Analog Scale (VAS) (Breivik et al., Br. J. Anaest. 2008 Jul; 101(1): 17-24). The validated measurement VAS was combined with a numerical scoring system, allowing the study to convert the data into a pain rating scale.

[0327] The weekly reporting section is designed to make it very simple for participants to give a score. The scoring tool used was selected based on the accuracy demonstrated in previous similar studies to track the subjective pain measurements of participants (e.g., Hawker et al. Arthritis Care Res. (Hoboken). 2011 Nov; 63 Suppl 11: S240-52, Kumar et al. J. Sci. Food Agric. 2015 Mar 15; 95 (4): 702-7). At the beginning of the study, each participant must define their baseline score for joint pain. Each week, participants were asked to indicate their pain score starting from Week 1 and throughout the trial until Week 16.

[0328] Participants also had to fill out a short demographic questionnaire asking about gender, age (within a certain range), height, weight, and waist measurement.

[0329] Participants' individual pain score combinations were anonymized in accordance with the General Data Protection Regulation (EU GDPR).

[0330] Calculate the weighted matrix for average activity impact

[0331] To assess the average impact of physical activity, a weighted matrix was used to classify participants according to the intensity of the activity (low (L), moderate (M) and high (H)) and the number of activities (1×, 2×, 3×). Walking, swimming, flexibility / balance, gardening, cycling, dancing and horseback riding were considered low joint-impact activities. Weightlifting, gym aerobics and boxing were considered moderate joint-impact activities. Running, ball games, skipping and heavy weightlifting were considered high joint-impact activities. The classification of activities was based on the National Health Service (NHS) guidelines (Physical Activity Guidelines for Older Adults). Available online: https: / / www.nhs.uk / live-well / exercise / physical-activity-guidelines-older-adults / #moderate (accessed on March 3). Regarding the number of activities, "LLL" means that the participant participated in three different types of low-intensity activities, "LLM" means that the participant participated in two different types of low-intensity activities and one type of moderate-intensity activity, and "LLH" means that the participant participated in two different types of low-intensity activities and one type of high-intensity activity. The same principle applies to the other subgroups. Given that only one participant met the MHH category (1× moderate and 2× high), and that alone would not be statistically relevant, this participant was considered to be in category 5 (LHH or MMH or MHH).

[0332] Statistical analysis

[0333] The full analysis population consisted of all eligible participants who took the supplement at least once and reported taking a VAS measurement after exercise at least once per week.

[0334] Continuous data were summarized according to their mean, standard deviation (SD), and range.

[0335] Categorical data were summarized by frequency and percentage.

[0336] A generalized linear mixed-effects model (LME) was used with a random intercept for each subject (b i The effect of treatment (HC vs. placebo) on the change in mean joint pain scores during follow-up compared to baseline was investigated in detail (each subject had its own intercept). Follow-up time was considered a factor variable, and since supplementation was discontinued at week 12, the analysis was based on the period from baseline until week 12.

[0337] The full model with interactions:

[0338] Y ij =(β0+b i )+β1 time ij +β2 external ij +β3 time ij ×Processing ij +ε ij

[0339] The interaction term represents the difference in expected joint pain change, comparing subjects from the HC group with those from the placebo group over a given follow-up period. In the case of a non-significant interaction term, it can be concluded that there is insufficient evidence of a difference between the treatment groups. A second analysis was then performed, which included only the main effects.

[0340] Systematic deviations from the normality assumption were verified using QQ plots, and systematic deviations from the mean model were examined using scatter plots of residuals versus predicted values.

[0341] LME analyses were performed in R (R Core Team, 2019) using the lmne package (Pinheiro et al. 2021. nlme: Linear and Nonlinear Mixed Effects Models. R package, version 3.1-153). All analyses were performed in R, version 3.6.0 (R Core Team, 2019). A p value < 0.05 was considered statistically significant. No imputation was performed for missing data. Tables present missing data where appropriate.

[0342] result

[0343] Subjects included in the study

[0344] A total of 213 subjects were enrolled in the study (HC / placebo: 177 / 36). The dropout rate in the placebo group was 19% (7 / 36) and the dropout rate in the HC group was 3% (5 / 177). Therefore, the application tool for follow-up was attractive to participants.

[0345] Object Characteristics

[0346] The demographic and baseline characteristics of the subjects, overall and by treatment group, are summarized in Table 2. The age range of the subjects was 18 to 72 years (mean age: 41 years), with 52.6% of the subjects being female.

[0347] Table 2. Demographic characteristics of subjects included in the study

[0348] variable Total (N=213) HC (N=177) Placebo (N=36) gender female 112(52.6％) 94(53.1％) 18(50.0％) male 101(47.4％) 83(46.9％) 18(50.0％) Age (years) Mean (SD) 40.7(11.0) 40.2(11.0) 42.1(11.0) scope 18-72 18-67 2372 Age category ＜30 39(18.3％) 35(19.8％) 4(11.1％) 30-39 59(27.7％) 48(27.1％) 11(30.6％) 40-49 69(32.4％) 57(32.2％) 12(33.3％) 50-55 27(12.7％) 22(12.4％) 5(13.9％) 55+ 19(8.9％) 15(8.5％) 4(11.1％)

[0349] Joint pain relief by HC vs placebo

[0350] Figure 1 The changes in joint pain in participants over a 16-week period are shown. A linear mixed effects model revealed a significant interaction between treatment and treatment duration (p < 0.001). The results indicate that there were differences between the placebo and HC groups in terms of subject-specific changes in expected joint pain sensation. When compared to subjects from the placebo group, subjects taking HC showed a significant (p < 0.001) reduction in mean joint pain sensation starting from week 3. It is also noteworthy that the difference between the HC and placebo groups increased with treatment duration.

[0351] After 12 weeks, treatment was stopped and the mean joint pain scores in the HC group began to increase again ( Figure 1 At week 16, the mean joint pain score in the HC group remained significantly lower than at baseline (paired sample t-test, p < 0.001), and there was also a significant difference between the HC group and the placebo group (independent sample t-test, p < 0.001). The reduction in joint pain persisted for 6 to 8 weeks after stopping treatment.

[0352] Figure 2 The effect of HC on joint pain compared to placebo is shown (by sex). There was no evidence of a significant three-way interaction, and there was no interaction between sex and treatment or between sex and time (p>0.05). In addition, the LME model revealed that joint pain scores were statistically similar between sexes (p=0.469).

[0353] Figure 3 The effect of HC on joint pain compared with placebo is shown (by age category). The LME model revealed a significant interaction between age and treatment time (p=0.005), emphasizing that for HC treatment, different age categories differed over time in terms of their joint pain levels. Therefore, using participants aged <30 years (whose joint pain scores were reported lowest in the age category) as a reference, the joint pain relief patterns between age categories treated with HC were compared. After 10 weeks of treatment, subjects aged 40 to 49 years had an average higher joint pain score compared with subjects aged <30 years (mean difference 0.71, 95% CI 0.25 to 1.18, p<0.01). This difference was recorded in the later stages of the study. Study participants less than 30 years of age reported the lowest joint pain scores between age categories. In general, across all age categories, all subgroups had joint pain levels that were significantly lower than the levels reported at the study baseline.

[0354] Figure 4The effects of HC compared to placebo on joint pain according to physical activity level are shown. Statistical analysis revealed that joint pain scores were consistent across physical activity subgroups (p=0.091). The extent of the treatment differences indicated that the effects of HC treatment were consistent across physical activity subgroups.

[0355] Overall, a reduction in joint pain was observed after HC intake, regardless of sex, age group, or activity level. This suggests that HC intake has a positive effect on a general population comprising active adults of varying age groups and lifestyles. The general trend was that a reduction in joint pain began to be observed 3 weeks after starting HC intake and persisted for 6 to 8 weeks after cessation of HC intake.

[0356] The reduction in joint pain was greatest in subjects in the age category <30 years; these are likely to be the most active and healthy subjects.

[0357] Table 3 shows the pain reduction by HC in subjects experiencing different types of joint pain. A reduction in joint pain was observed regardless of the nature of the joint pain, but the effect was most pronounced in (healthy) subjects after exercise.

[0358] Table 3. Changes in VAS scores in subjects experiencing different types of joint pain after daily oral administration of HC for 12 weeks

[0359] Types of joint pain Decreased VAS score Joint pain in septic arthritis lowest Joint pain in osteoarthritis Average / High Joint pain after exercise Highest

[0360] Example 3

[0361] The method of Example 1 was used, wherein pig bones, pig skin, pig trachea or pig sternum were compared as starting materials for the hydrolysis.

[0362] Table 4 shows that daily treatment with compositions derived from pig bones or pig skin did not result in a significant reduction in joint pain after 12 weeks. In contrast, daily treatment with collagen hydrolysate compositions derived from pig trachea or pig sternum resulted in a reduction of more than 50% in VAS scores after 12 weeks. Similar results were obtained with bovine-derived tissue. Chicken sternum was found to be less effective in reducing joint pain compared to pig or bovine sternum, suggesting that there may be species dependence in the pain-relieving effect. Table 4. VAS scores after 12 weeks of daily intake of HC derived from different collagen-containing substances.

[0363] VAS score Placebo 4.5 HC from pig bones 4.0 HC from pigskin 4.0 HC from pig trachea 2.0 HC from pig sternum 2.0

[0364] Example 4

[0365] The method of Example 1 was used, but using different combinations of porcine bones, porcine skin, porcine trachea, and porcine sternum to obtain starting materials for hydrolysis having different relative amounts of collagen types I, II, and III.

[0366] Table 5 shows that compositions prepared from starting materials containing primarily type I or type II collagen have a pain-relieving effect, albeit a limited one. In contrast, compositions prepared from starting materials containing type I, II, and III collagen in a weight ratio of approximately 5:5:1 have a much greater pain-relieving effect. The collagen hydrolyzate obtained from this starting material has a Hyp / Hyl molar ratio greater than 8 and less than 15.

[0367] The pain-relief effect is greatest when the amount of chondroitin sulfate in the composition is relatively high. In this example, a composition containing 70% collagen hydrolysate and 30% chondroitin sulfate was found to provide the greatest pain relief. Table 5. Starting materials, resulting compositions, and pain-relief effects. HC: collagen hydrolysate, CS: chondroitin sulfate, Hyp / Hyl: molar ratio of hydroxyproline to hydroxylysine.

[0368]

[0369] Table 6 shows that type III collagen contributes to pain relief. A ten-fold reduction in type III collagen in the starting material resulted in a lower pain relief effect.

[0370] Table 6. Starting materials, obtained compositions and pain-relieving effects. HC: collagen hydrolysate, CS: chondroitin sulfate, Hyp / Hyl: molar ratio of hydroxyproline to hydroxylysine.

[0371]

Claims

1. A composition for improving joint pain after exercise in a subject, The composition comprises 10% to 40% by weight of chondroitin sulfate and 60% to 90% by weight of collagen hydrolysate, calculated based on the total weight of the chondroitin sulfate and the collagen hydrolysate. The collagen hydrolysate contains hydroxyproline and hydroxylysine, and the molar ratio thereof is greater than 8 and less than 15.

2. The composition of claim 1, wherein the subject is a healthy individual.

3. The composition according to claim 1 or 2, wherein the collagen hydrolysate comprises 2 wt% to 20 wt% hydroxyproline, wherein the wt% is calculated based on the total weight of amino acids in the collagen hydrolysate.

4. The composition according to claim 1 or 2, wherein the collagen hydrolysate comprises 0.1 wt% to 5 wt% hydroxylysine, wherein the wt% is calculated based on the total weight of amino acids in the collagen hydrolysate. 5 . The composition according to claim 1 , comprising 15 to 40 wt % of chondroitin sulfate, calculated based on the total weight of the chondroitin sulfate and the collagen hydrolyzate. 6 . The composition according to claim 1 , comprising 20 to 40 wt % of chondroitin sulfate, calculated based on the total weight of the chondroitin sulfate and the collagen hydrolyzate. 7 . The composition according to claim 1 , comprising 30 to 40 wt % of chondroitin sulfate, calculated based on the total weight of the chondroitin sulfate and the collagen hydrolyzate.

8. The composition according to claim 1 or 2, wherein the average molecular weight of the collagen hydrolysate is greater than 1000 Da and less than 10000 Da.

9. The composition according to claim 1 or 2, wherein the composition is administered orally.

10. The composition according to claim 1 or 2, wherein the composition is administered in a daily dose of 0.2 to 2 g, wherein the daily dose is the total dry weight of the composition.

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